Outbreak of Pandoraea commovens Infections among Non–Cystic Fibrosis Intensive Care Patients, Germany, 2019–2021

Pandoraea spp. are gram-negative, nonfermenting rods mainly known to infect patients with cystic fibrosis (CF). Outbreaks have been reported from several CF centers. We report a Pandoraea spp. outbreak comprising 24 non-CF patients at a large university hospital and a neighboring heart center in Germany during July 2019–December 2021. Common features in the patients were critical illness, invasive ventilation, antimicrobial pretreatment, and preceding surgery. Complicated and relapsing clinical courses were observed in cases with intraabdominal infections but not those with lower respiratory tract infections. Genomic analysis of 15 isolates identified Pandoraea commovens as the genetically most similar species and confirmed the clonality of the outbreak strain, designated P. commovens strain LB-19-202-79. The strain exhibited resistance to most antimicrobial drugs except ampicillin/sulbactam, imipenem, and trimethoprim/sulfamethoxazole. Our findings suggest Pandoraea spp. can spread among non-CF patients and underscore that clinicians and microbiologists should be vigilant in detecting and assessing unusual pathogens.

Pandoraea spp.are gram-negative, nonfermenting rods mainly known to infect patients with cystic fibrosis (CF).Outbreaks have been reported from several CF centers.We report a Pandoraea spp.outbreak comprising 24 non-CF patients at a large university hospital and a neighboring heart center in Germany during July 2019-December 2021.Common features in the patients were critical illness, invasive ventilation, antimicrobial pretreatment, and preceding surgery.Complicated and relapsing clinical courses were observed in cases with intraabdominal infections but not those with lower respiratory tract infections.Genomic analysis of 15 isolates identified Pandoraea commovens as the genetically most similar species and confirmed the clonality of the outbreak strain, designated P. commovens strain LB-19-202-79.The strain exhibited resistance to most antimicrobial drugs except ampicillin/sulbactam, imipenem, and trimethoprim/sulfamethoxazole.Our findings suggest Pandoraea spp.can spread among non-CF patients and underscore that clinicians and microbiologists should be vigilant in detecting and assessing unusual pathogens.
Pandoraea spp.colonization and clinical deterioration is not always clear (9,11,12).The potential of Pandoraea spp. to cause acute illnesses has been exemplified by bloodstream and other life-threatening infections in CF patients and patients who received solid organ transplantation (7,(13)(14)(15).
Pandoraea outbreaks have been documented in CF centers in Denmark and France, each comprising 6 patients (9,12).One in-depth analysis described a large P. apista cluster affecting 18 CF patients serviced at the pediatric and adult CF centers in a city in Scotland and 1 other patient from south England (6).We report a Pandoraea spp.outbreak during July 2019-December 2021 at a large university hospital and the directly neighboring heart center in Berlin, Germany, involving 24 non-CF patients colonized or infected with a novel P. commovens strain.

Patient Data
We retrospectively extracted patient data from hospital records.Data included length of hospital stay, time to isolate Pandoraea spp., antimicrobial drug treatment, intensive care unit (ICU) admission, renal dialysis, solid organ transplantation, underlying conditions exemplified by the Charlson Comorbidity Index (CCI) scores, and patient outcome.We classified patients as either colonized or infected according to the judgment of 2 infectious disease consultants.Detection of Pandoraea spp.from otherwise sterile sites, such as blood, or from intraabdominal specimens was considered as infection.Culture from nonsterile sites (e.g., respiratory samples) was considered colonization if further assessment of antibiotic prescriptions, physicians' notes, laboratory values, and radiology and pathology findings did not reveal evidence of infection.For the diagnosis of pneumonia, >1 of the following criteria had to be met: new or progressive infiltrate, new or worsening respiratory signs and symptoms, or rising inflammatory markers and assessment of pneumonia by the treating physician.For difficult cases, 2 clinicians discussed and then agreed on a classification for each case (Appendix, https://wwwnc.cdc.gov/EID/article/29/11/23-0493-App1.pdf).
We also conducted environmental investigations for a point source of P. commovens.For environmental investigations, we probed respiratory tubes, nebulizers, suction catheters, washing gloves, toothbrushes, nutrition solutions, inhalation solutions, oral medications such as painkillers in solution, eye and nasal ointments, and eye drops.

Genomic Sequencing and Analysis
For exact species identification and determination of clonality, we subjected 15 clinical outbreak isolates to WGS, by using either the Nextera Flex (Illumina, https://www.illumina.com)or QIASeq FX (QIAGEN, https://www.qiagen.com)library preparation kits, according to the manufacturers' protocols.In brief, we extracted and enzymatically fragmented 10-100 ng of DNA by using DNeasy PowerSoil Pro Kit (QIAGEN).We added indexed adapters and amplified libraries in limited-cycle PCRs.After clean-up, we quantified, normalized, and pooled sequenceready libraries before sequencing by using 2× 250 cycles paired-end sequencing on a MiSeq (Illumina).
To close the genome, we performed nanopore sequencing on genomic DNA from isolate LB-19-202-79 from our outbreak on GridION (Oxford Nanopore Technologies, https://nanoporetech.com) using an R9.4 flow cell (Oxford Nanopore).We prepared the sequencing library by using the SQK-LSK109 Ligation Sequencing Kit (Oxford Nanopore), according to the manufacturer's protocol.We performed basecalling by using Guppy version 5.0.11(Oxford Nanopore) on the SUP accuracy setting in GridION.
We screened the genome assembly of LB-19-202-79 against all available Pandoraea spp.assemblies in the NCBI RefSeq database (29) as of July 21, 2022, by using mash-screen version 2.3 (30).We calculated average nucleotide identity (ANI) between genome assemblies by using FastANI version 1.33 (31).We used andi version 0.12 (32) to calculate pairwise genetic distances between the assembled genomes, from which we constructed a phylogenetic tree comprising the isolate assemblies and the closest Pandoraea spp.by using the neighbor joining method of the ape package version 5.6 (33).
We uploaded the genome assembly to GenBank for annotation by using the NCBI Prokaryotic Genome Annotation Pipeline (34).The uploaded assembly was then automatically annotated by the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) pipeline (https://www.bv-brc.org).

Ethics, Consent, and Permissions
The study was approved by the internal review board of Charité-Universitätmedizin Berlin (registry no.EA4/145/21).The need for informed consent was waived because the study was retrospective.

Results
During July 2019-December 2021, we registered phenotypically identical Pandoraea spp.isolates in specimens from 24 patients, which is 8 times the number of all Pandoraea spp.detected at our laboratory in the 3 previous years (2016-2018).The cases clustered at Charité Campus Virchow Klinikum (CVK) and Deutsches Herzzentrum Berlin (DHZB), 2 neighboring institutions that are on the same grounds; staff and patients regularly move between the 2 institutions.Thirteen patients were treated at DHZB and 9 were treated at CVK.One other patient was treated at Charité Campus Benjamin Franklin and 1 at Unfallkrankenhaus Berlin, a major trauma center; both of those institutions are in different districts of the city.A total of 7 ICUs, 3 at DHZB and 4 at Charité CVK, and 3 regular wards were affected by the outbreak.Cases were first observed at Charité CVK, then the outbreak shifted after a patient was transferred to 2 ICUs at DHZB.Since late August 2019, nearly all isolates have been recovered on those 2 ICUs (Figure 1).Environmental investigations performed at those ICUs in September 2019 did not reveal any point source.

Patient Information and Outcomes
Among the 24 patients whose cultures grew Pandoraea spp., the median age was 67 (range 45-81) years; 50% were male and 50% female.Clinical data were available on 23 patients.Median time from admission to Pandoraea spp.detection was 22 days; 22 (96%) of the 23 patients were treated in ICUs.Patients had numerous underlying conditions, and the median CCI was 6 (range 0-13).All patients had received antimicrobial drug treatment during their hospital stays before Pandoraea spp.detection, 65% had undergone surgery, and 65% received mechanical ventilation (Table 1).
We considered 12 patients colonized and 10 patients infected.For 2 cases, we were unable to make a classification.All 10 of the infected patients were on mechanical ventilation, compared with only 4 (33%) colonized patients.Among infected patients, 8 had lower respiratory tract infections and 2 had intraabdominal infections.In 5 infected patients, Pandoraea spp. was part of a polymicrobial culture, and in the other 5 infected patients, no other pathogens were detected.Pandoraea spp.infections were treated with imipenem in 8 patients and meropenem in 2 patients.TMP/SMX was administered as stepdown therapy in 1 patient after initial treatment with imipenem.All respiratory tract infections resolved, whereas the 2 patients with intraabdominal infections had complicated clinical courses that involved several surgical interventions and protracted administration of various antimicrobial agents in both cases (Appendix).

Microbiology Results
During July 2019-December 2021, Pandoraea spp. was cultured from 43 clinical specimens, including throat swabs, respiratory secretions, bile, ascites, intraabdominal specimens, and wound swabs.Numerous blood cultures were collected from 19 of the 24 patients.However, Pandoraea spp. was only detected in 1 blood culture from a patient with a complicated intraabdominal infection.Pandoraea spp.isolates grew readily after overnight culture on commercial solid media, such as Columbia blood or MacConkey agars.Single colonies appeared pale to grayish, displayed weak oxidase activity, and were catalase negative.Neither a mucoid phenotype nor small colony variants were observed.
Using the VITEK 2 GN ID card, identification was possible only to the genus level.In 4 of 17 tested isolates, a reliable discrimination between Pandoraea spp.and Bordetella hinzii could not be made by VITEK 2. In the remaining 13 isolates, we identified Pandoraea spp. with probabilities ranging from 95% to 99%.All 36 isolates tested by VITEK MALDI-TOF mass spectrometry were identified as P. sputorum with a score of 99.9.We were able to perform WGS to differentiate P. commovens from P. sputorum on isolates from patients 7, 9-11, and 14-24, as described in the next section.Because of the local and temporal relationship, identical colony morphology and antimicrobial susceptibilities, we assumed that the correct species identification was P. commovens in all patients reported in this outbreak and that P. sputorum was a misidentification resulting from limitations in the VITEK mass spectrometry database.
We performed susceptibility testing on 35 isolates.For susceptibility testing methods, agreement between VITEK 2 and MICRONAUT-S broth microdilution plates was good for most tested antimicrobial agents.According to EUCAST PK/PD breakpoints, MICs obtained for ampicillin/sulbactam and imipenem were consistently found to be susceptible at standard dosing levels.MICs for TMP/SMX were <20 mg/L in all tested isolates.Cephalosporins including ceftazidime/ avibactam, ceftolozane/tazobactam, and cefiderocol tested resistant, as did fluoroquinolones, aminoglycosides, tetracyclines, and colistin (Table 3).
Discrepancies between VITEK 2 and microdilution were apparent for piperacillin and piperacillin/ tazobactam; higher MICs were detected using VITEK 2 (range 16 to >128 mg/L for both) than with microdilution (range <4 to 32 mg/L for piperacillin and <1 to 2 mg/L for piperacillin-tazobactam).MICs for meropenem and ertapenem were also higher in VITEK 2 (range 4-8 mg/L and 1 to >8 mg/L, respectively) than in microdilution (range 1-4 mg/L and <0.5 to 1 mg/L, respectively).

Genomic Characterization and Phylogeny
We screened the genome assemblies from 15 isolates from our outbreak against genomes in RefSeq.We found the genome assembly GCF_902459615.1 of P. commovens strain LMG 31010 to be the most similar, then P. sputorum strain ATCCBAA64, and P. oxalativorans strain DSM 23570.The average genome-wide nucleotide identity between isolate LB-19-202-79 and GCF_902459615.1 was 99.5% and identity between LB-19-202-79 and P. sputorum GCF_900187205.1 was 94.1%.Thus, we designated our strain as P. commovens strain LB-19-202-79.The phylogenetic tree derived from the pairwise phylogenetic distances showed that all our isolates are closely related to each other and distinct from P. commovens strain LMG 31010 and other Pandoraea spp.(Figure 2).We concluded that the outbreak isolates were from a single origin.
A search of the plasmid sequence against the NCBI BLAST nucleotide database (https://blast.ncbi.nlm.nih.gov)revealed several alignments to the plasmid of Burkholderia aenigmatica strain CMCC(B)23010 (GenBank accession no.CP091649.1),totaling ≈22 kb and ≈99.9% sequence identity.That strain is a member of the Burkholderia cepacia complex and was originally isolated from water purified for pharmaceuticals.Apart from that, we only found alignments to transposase genes in Klebsiella pneumoniae plasmids.
The annotation of the LB-19-202-79 chromosome yielded 2 β-lactamase family proteins that are also found in the P. commovens strain LMG 31010 genome assembly and have >99% amino acid identity.The β-lactamase with locus tag NTU39_20675 was identified as an oxacillinase (OXA) 62 family carbapenem-hydrolyzing class D enzyme and now is denoted as allele blaOXA-1149 in the NCBI Reference Gene Catalog.The β-lactamase with locus tag NTU39_00730 was identified as a class C β-lactamase.We analyzed the complete resistome comprising all genes associated with antimicrobial esistance as determined by the BV-BRC genome annotation (Appendix Table ).The complete genome annotation is available at BV-BRC (accession no.2508289.5;https://www.bv-brc.org/view/Genome/2508289.5).

Discussion
We describe a large Pandoraea spp.outbreak comprising 24 non-CF patients.In contrast to earlier outbreaks that took place uniformly among CF patients (6,9,12), none of the patients in this outbreak had CF.However, all but 1 patient were treated in an ICU immediately before or during the time when P. commovens was isolated.All patients had received antimicrobial drugs before P. commovens isolation, and all likely acquired the pathogen in the hospital.Most of the patients had undergone surgery or were on mechanical ventilation, and their overall CCI was high (median 6, range 0-13).Those observations align with earlier case reports on Pandoraea spp.infections among non-CF patients (16)(17)(18)(19)(20)(21).
As described by others (35), we experienced difficulties in correctly identifying the species of the outbreak strain.In several cases, P. commovens was misidentified as B. hinzii by biochemical means.Because P. commovens was not identified as a separate species before November 2019 (2), VITEK MALDI-TOF mass spectrometry analysis misidentified the outbreak strain as P. sputorum.
Pandoraea spp.can harbor multiple antimicrobial resistance and biodegradation genes, enabling the pathogen to persist in the hospital environment (6).Our resistome analyses and the course of this outbreak that lasted for 2.5 years suggest that P. commovens LB-19-202-79 is equipped with such an armament.
Pandoraea spp.exhibit resistance to most antimicrobial agents, including penicillins, cephalosporins, fluoroquinolones, aminoglycosides, and colistin, but frequently are susceptible to imipenem and TMP/SMX (14,36,37).Resistance is mediated by different efflux pumps and β-lactamases with a carbapenem-resistant phenotype observed in isolates carrying OXA-62 or a homologue to OXA-153, both carbapenem-hydrolyzing oxacillinases.OXA-62 hydrolyzes meropenem more efficiently than imipenem, but expanded-spectrum cephalosporins are only poor substrates (5,6,38).Among the β-lactamase family proteins detected in our strain, one was identified as an OXA-62 family carbapenem-hydrolyzing class D β-lactamase, now denoted as OXA-1149.However, all our P. commovens isolates were susceptible to imipenem but showed elevated MICs (1-8 mg/L) for meropenem, corresponding to susceptible to increased exposure according to EUCAST PK/PD non-species related breakpoints.The high-level resistance of P. commovens to all cephalosporins might at least in part be mediated by the expression of a class C β-lactamase (39).Although all 8 patients with respiratory tract infections recovered with a single course of antimicrobial drugs, the 2 patients with P. commovens intraabdominal infections had relapsing courses of disease.Pandoraea spp.are environmental bacteria and can thrive in wet settings, such as an abdominal area undergoing multiple surgeries.Under such circumstances, armed with a class D carbapenemase, P. commovens LB-19-202-79 might withstand even prolonged targeted antimicrobial treatment, as noted in patient 7 (Appendix).
The assessment of the clinical significance of detection of P. commovens in the patients in this outbreak was not always straightforward, especially in cases where Pandoraea spp. was cultured from respiratory secretions.Some cases could easily be classified as colonization, such as when only throat swab samples were positive and patients had no other signs and symptoms of infection.However, P. commovens was part of polymicrobial cultures in some patients, and its role in those cases was difficult to estimate.P. commovens was the only relevant pathogen that could be detected in 5 of 8 patients with nosocomial pneumonia; however, it was only detected in low to intermediate quantities.Those cases were classified as infections because patients had nosocomial pneumonia, which cleared after administration of targeted antimicrobial drug treatment for P. commovens.The pathogenicity of P. commovens in the 2 patients with complicated intraabdominal infections seemed evident.P. commovens was the predominant pathogen and was repeatedly isolated from different materials, even from blood culture in 1 case.
The first limitation of this study is that we were not able to sequence all Pandoraea spp.isolates from the outbreak.Some uncertainty remains about whether all isolates belonged to the outbreak strain P. commovens LB-19-202-79.However, given the identical phenotypic features and the temporal and spatial relationship, we assume the same strain was responsible for all cases.Second, we were not able to find an environmental source.Third, detection of Pandoraea spp.could not easily be classified as colonization or infection in several patients; however, that is a well-known dilemma when low-virulent pathogens are cultured from nonsterile sites, such as the respiratory tract.
In conclusion, our sequence analysis highlights the advantage of bacterial WGS for exact species identification and typing of outbreak isolates.On the basis of these findings, we conclude that Pandoraea spp.are not only capable of spreading among CF patients, as described before, but also to non-CF patients.The bacteria can also cause outbreaks on ICUs, in particular affecting patients with a history of intensive antimicrobial pretreatment, multiple abdominal surgeries, and mechanical ventilation.This outbreak report underscores the critical role of vigilance among both clinicians and microbiologists when unusual pathogens occur and the need for access to modern molecular sequencing techniques in hospital laboratories.
All sequencing data are available from the NCBI under BioProject no.PRJNA849608.Byron Breedlove, managing editor of the journal, elaborates on aesthetic considerations and historical factors, as well as the complexities of obtaining artwork for Emerging Infectious Diseases.

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Figure 1 .
Figure 1.Timeline of an outbreak of Pandoraea commovens among non-cystic fibrosis intensive care patients, Germany, 2019-2021.The cases clustered at Charité Campus Virchow Klinikum (university hospital) and Deutsches Herzzentrum Berlin (heart center), 2 neighboring institutions that are on the same grounds and staff and patients regularly move between the 2 institutions.Wards 1-7 are located at the same grounds; wards 8 and 10 are located in facilities elsewhere in Berlin (indicated by grey background).X symbols indicate calendar weeks with detection of P. commovens.;horizontal bars in timelines indicate length of stay.ICU, intensive care unit; ND not done; Pt., patient; WGS ID, whole-genome sequencing identification number corresponding to the numbering in phylogenetic tree (Figure 2).

Figure 2 .
Figure 2. Phylogenetic tree of isolates from an outbreak of Pandoraea commovens among non-cystic fibrosis intensive care patients, Germany, 2019-2021.Genome assemblies from 15 isolates (labeled LB) compared with Pandoraea spp.genomes in the National Center for Biotechnology Information RefSeq database (https://www.ncbi.nlm.nih.gov)found the genome assembly GCF_902459615.1 of P.commovens strain LMG 31010 was the most similar.The tree was created by using neighbor joining the calculated pairwise phylogenetic distances between genome assemblies and available database sequences.Scale bar indicates nucleotide substitutions per site.

Table 1 .
Patient characteristics in an outbreak of Pandoraea commovens among non-cystic fibrosis intensive care patients, *Data reflect 23 cases, except for sex, median age, and median length of hospitalization (n = 24).CKD, chronic kidney disease.†Values are no.(%) or median (range).